Targeted disease prophylaxis in European fish farming

Final Report Summary - TARGETFISH (Targeted disease prophylaxis in European fish farming)

Executive Summary:
Aquaculture production in Europe is responsible for the employment of 100,000 people, generating an annual turnover of 7 billion EUR. However, partly due to a lack of authorised veterinary products for medicinal treatment, the consequential outbreaks of disease in farmed fish species can cost the sector up to 20 % of its production value. The most appropriate method for controlling the spread of disease is to prevent it from starting in the first place, through vaccination.

TARGETFISH aimed to provide a long-lasting contribution to the prevention of important fish diseases in the European aquaculture industry by developing fish vaccines against several bacterial and viral pathogens. Specifically, its research focused on the generation of knowledge on bacterial and viral proteins (i.e. antigens), new oral systems for delivering these antigens to mucosal body sites including the gut, and new adjuvants for improving the duration of immunity. This information was then evaluated against both mucosal and systemic protective immune responses. Researchers next turned their attention to learning from and improving upon, existing vaccines and focused on prototyping vaccines whose efficacy was then validated via in vitro assays and in vivo challenges. Successful vaccines were evaluated for minimal side effects and maximum safety. Lastly, some of the novel vaccination protocols were scrutinised under field conditions, shortening the route for the implementation of the project’s results. Thoughout the project, results were presented to policy makers, scientists and industry leaders.

During the course of the work, TARGETFISH researchers came up against several challenges that required innovative solutions. For example, although DNA vaccination by injection had already been shown to be effective in the laboratory, its application in Europe had been halted due to debates around the safety of DNA-based vaccines. While several research groups confirmed the efficacy of this form of vaccination, TARGETFISH placed substantial effort on studying the genome integration aspect, producing data on the integration of plasmid DNA in muscle tissue of vaccinated fish. To date, DNA vaccination may rapidly become a reality in Europe. TARGETFISH also specifically aimed to integrate the many SME partners who provided improved forms of antigens, vaccination and delivery methods. One important issue addressed with the help of these enterprises was the development of oral vaccines that protect antigens from degradation in the gastro-intestinal tract. Although frequently a strong uptake of antigen and correlated immune responses in the gut could be measured, oral vaccination did not always lead to protection when the fish were challenged. Yet, there is clear hope to see improved protection in the future when doses and duration of feeding oral vaccines are optimised.

Overall, TARGETFISH has been revolutionary in that it not only generated fundamental but strategic knowledge for the development of next generation fish vaccines and different routes of vaccine administration, but it also validated this knowledge by actively working on rapid implementation of improved or new prototype fish vaccines.

Project Context and Objectives:
Aquaculture production in Europe is responsible for the employment of 100,000 people, generating an annual turnover of 7 billion EUR. However, partly due to a lack of authorised veterinary products for medicinal treatment, the consequential outbreaks of disease in farmed fish species can cost the sector up to 20 % of its production value. The most appropriate method for controlling the spread of disease in farmed fish species is to prevent it from starting in the first place, through vaccination. The EU-funded TARGETFISH project set out with one main objective: to effectively vaccinate fish.

TARGETFISH strived to advance the development of existing (but not sufficient) and new prototype vaccines against socio-economically important viral or bacterial pathogens of Atlantic salmon, rainbow trout, common carp, sea bass, sea bream and turbot (Table 1).

Table 1. Overview of fish species addressed by the TARGETFISH project and pathogens targeted for vaccine development

Fish species Disease
Atlantic salmon IPN Infectious Pancreatic Necrosis
PD Pancreas Disease
Rainbow trout RTFS Rainbow Trout Fry Syndrome
ERM Enteric RedMouth
VHS Viral Hemorrhagic Septicemia
Turbot vibrio Vibrio anguillarum
Carp SVC Spring Viremia of Carp
KHV Koi HerpesVirus
Sea bass VER Viral Encephalopathy and Retinopathy
Seabream Pasteurellosis Photobacterium damselae subsp. piscicida

The project aimed to develop targeted vaccination strategies and bring improved vaccines closer to industrial application by also addressing practical issues such as efficacy, safety and delivery route. To achieve these challenging tasks for as many as six different fish species and ten different fish diseases, TARGETFISH brought together 30 partners from 11 EU member states, 2 associated countries and 1 International Cooperation Partner Country (ICPC). In this large multidisciplinary consortium an approximate equal number of RTD and SME partners cooperated closely while keeping an intensive communication with the large vaccine and nutrition industries via an Industry Forum.

Specifically, TARGETFISH aimed to establish a knowledge- and technology-base for rational development of next generation fish vaccines and enhance targeted disease prophylaxis in European fish farming by:

1) generating knowledge on antigens and adjuvants for systemic as well as mucosal routes of administration while analyzing the underpinning protective immune mechanisms;
2) validating this knowledge with immune response assays for monitoring vaccine efficacy and study safety aspects, including those associated with DNA vaccines;
3) approaching implementation of prototype vaccines by optimizing vaccination strategies thus shortening the route to exploitation.

Traditionally, many fish vaccines have been based on inactivated bacteria or viruses, and indeed several of these have shown extremely (cost) effective. But not all fish pathogens can be fought with these relatively simple type of vaccines and sometimes next generation (subunit, recombinant) vaccines are needed. However, these next generation vaccines can sometimes have lower immunogenicity than have vaccines with complete pathogens, thus specific adjuvants must be found suitable to combine with the antigen to reach significant protection levels. DNA vaccines are based on administering a DNA sequence coding for the target antigen: the DNA is injected in the host, transcribed and the protein produced by the host itself, and can be highly efficacious but, similar to live attenuated vaccines, safety concerns can limit their practical use. TARGETFISH not only studied new antigens but also studied new formulations of DNA plasmids to provide objective information on safety and efficacy with the aim to facilitate future decision making on the implementation of highly-effective DNA vaccines.

Vaccine antigens can be produced by traditional methods, including inactivation of bacteria and viruses, or produced in bulk culture for example when expressed as recombinant products in bacterial or yeast expression systems. The yeast Pichia pastoris has a high growth rate and can grow on a simple, inexpensive medium in either shake flasks or a fermenter, making it suitable for large-scale production of antigens for fish vaccines. Under optimal conditions, Pichia can grow to very high cell densities and produce recombinant proteins with the required disulfide bonds and glycosylations patterns at very high protein yields. TARGETFISH aimed to develop, for example, prototype Pichia-based vaccines against important viral diseases.

Most fish vaccines in the market are delivered by injection. Although this can be a labour-intensive, expensive method that can also provoke stress in these fish, under several circumstances injection vaccination has been the most effective way of vaccination, at least with respect to levels of protection. Since injection by hand is time consuming, automated vaccinating machines may help achieve mass injection vaccination. Of extreme importance is that such machines are designed properly to guide the fish into the machine correctly, so that injections are placed correctly in the intraperitoneal cavity. Minimal size requirements for the to-be-injected fish have been a limiting factor for the application of these machines. TARGETFISH aimed to assist with the development of automated vaccinating machines for injecting small fish of a few grams only, allowing for injection vaccination of juvenile fish.

Using mucosal vaccination routes (immersion or oral) is more desirable than using injection vaccination routes, but their optimization required more research. TARGETFISH focused on investigating mucosal vaccination routes and associated immunity and on developing antigens and adjuvants specifically suited for mucosal delivery. Microencapsulation includes the coating of vaccine antigens with specially selected materials such as alginates and should improve stabilisation of the vaccine antigens with respect to storage and facilitate that antigens reach the site of action (e.g. the local immune system in the gut). Alginates provide natural, biodegradable and muco-adhesive microencapsulation and can help reduce degradation in the acidic environment of the gastro-intestinal tract. Alginates are considered safe and can provide controlled-release of encapsulated protein antigen to target delivery to antigen-presenting cells of the local immune system and thus could assist the inclusion of antigens in oral vaccines. TARGETFISH aimed to enhance the use of alginates for fish vaccines by studying, for example, the best size of alginate particles which should be proportionate to the loaded protein (antigen) and administration route and indeed, the interaction between alginate particles and exact vaccine antigens may determine the final product. TARGETFISH aimed to make use of alginate microencapsulation of proteins from important fish bacteria and/or viruses to assist the formulation of next generation fish vaccines.

Overall, TARGETFISH addressed several different delivery methods for fish vaccines as promising new methods for mass vaccination, ranging from automated vaccination to oral delivery. It is known that vaccine antigens often require protection against the harsh environment of the stomach and/or gut and therefore the project addressed among others studies on alginates to help achieve oral delivery of vaccine antigens in a biologically-safe manner. Also, water-in-oil emulsions of antigen and adjuvant known to work efficiently for injection vaccines can sometimes trigger local inflammation and may not always provide the optimal formulation for oral vaccines, e.g. those delivered by alginates to the fish gut, for which reason alternative adjuvant formulations received special attention. And last but not least, since memory formation and development of specific immunity are central to successful vaccination of fish, TARGETFISH aimed to apply the most current knowledge of three antibody types recently identified in fish by measuring these specific antibodies after injection, oral or immersion vaccination. Thereby, TARGETFISH continued to develop fundamental knowledge for next generation fish vaccines and for application of different routes of vaccine administration, while continuously translating the newly-acquired fundamental knowledge into strategic knowledge by actively involving the vaccine industry.

Project Results:
TARGETFISH kicked off with 30 partners from 10 EU member states, two associated countries (Norway, Israel) and one International Cooperation Partner Country (Chile), with the aim to improve fish vaccination strategies to help prevent important diseases in the European aquaculture industry. Characteristic of TARGETFISH has been the close cooperation between academic research groups and (small to medium size) enterprises, both more or less equally represented in the consortium. The project brought together researchers on fish pathology and immunology who all shared one main interest: fish vaccination. The ambitious consortium chose to target vaccination against more than 10 important viral or bacterial pathogens of Atlantic salmon, rainbow trout, common carp, sea bass, seabream and turbot with the overall aim of advancing the development of existing but sometimes insufficient or suboptimal vaccines and develop new prototype vaccines. Here, a number of ‘case studies’ from the project are discussed.

Bacterins have proven and are still proving a good basis for excellent vaccines

Rainbow trout fry syndrome (RTFS) caused by one of the most devastating and pathogenic bacteria Flavobacterium psychrophilum is affecting the European rainbow trout industry. Prior to TARGETFISH the success of experimental vaccines against this disease had been limited because experimental vaccines provided only poor levels of protection upon experimental challenge. Sometimes for bacterial pathogens, specific antibody responses may be sufficient to confer protection, so TARGETFISH tested if vaccines composed of these inactivated microbes could be enough to trigger systemic reactions adequate for protection against RTFS. Bacterin vaccins typically are produced by inactivating the bacteria (and their products) through agents such as formalin, while assuring that the antigenic fragments of the proteins against which the antibody responses are directed remain intact. TARGETFISH built on the knowledge that these nonself molecules should be recognized by antibodies (or, immunoglobulins (Ig)) present on the cell surface of B lymphocytes, which then differentiate into antibody-secreting plasma cell factories. Because typically, B lymphocytes have evolved to recognize a great variety of different antigens we first studied the bacterial pathogen and thus antigen variability for F. psychrophilum.

TARGETFISH found a significant strain variability among >300 isolates of F. psychrophilum, and chose to use three representative isolates to produce a trivalent vaccine which was first administered by intra-peritoneal injection (with an oil-based adjuvant). The first trials indicated very good protection of rainbow trout upon experimental challenge >500 degree days both with a homologous and a heterologous bacterial strain. Subsequent trials in Atlantic salmon indicated protection after even longer periods of > 1000 degree days, whereas immersion rather than injection vaccination protocols followed by bath challenges of rainbow trout indicated that also with more practical (mass) vaccination routes for small sized fish, such as immersion vaccination, the use of bacterins to develop vaccines against bacterial diseases such as RTFS can be highly effective (Figure 1).

Figure 1. Percentage survival after bath challenge of replicate vaccinated and control groups of rainbow trout upon controlled infection with F. psychrophilum.

Adjuvants (also non oil-based) are key to help achieve protective immune responses

Presently several existing efficacious vaccines against bacterial diseases of fish have been based simply on empirically-optimised combinations of antigen and adjuvant, with one of the requirements for the adjuvant being that it allows for antigen persistence within the body cavity (at least when injected intra-peritoneally) to ensure optimal activation of the immune system. Another important requirement for the adjuvant is to help trigger innate immune responses; inducing expression of co-receptors necessary for an efficient activation of adaptive immunity and inducing long-lasting protection. Oil-based adjuvants generally meet both of these requirements but can also be associated with side effects owing to local inflammation and adhesions and may thus interfere with consumer acceptance and animal welfare. Thus, the use of specific molecules as alternative, non oil-based, adjuvants in vaccine formulations is of high interest. Similar to oil-based adjuvants, such “molecular” adjuvants should improve antigen persistence and uptake at the site of administration and promote the recruitment of immune cells to trigger the generation of antigen-specific memory cells. Ideally, these novel adjuvants not only induce limited or no side-effects but also ensure a targeted and efficient activation of both, innate and adaptive immune mechanisms.
Toll like receptors (TLRs) are a family of pattern recognition receptors (PRRs) that function as primary sensors of the innate immune system to recognize distinct structures in microbes such as pathogenic bacteria and viruses, these structures often referred to as “PAMPs” (pathogen associated molecular patterns) (Figure 2). Binding of such PAMPs to Toll like receptors invokes a cascade of intracellular signaling pathways that induce the production of inflammatory and thus stimulatory innate immune responses.

Figure 2. Sensing of Pathogen Associated Molecular Patterns (PAMPs) by Pattern Recognition Receptors (PRRs) present on cell membranes can enhance innate immune responses and thus have potential for boosting adaptive immune responses to fish vaccines.

Many Toll like receptors are expressed on the surface of (innate immune) cells to detect pathogens (often bacteria) within their immediate local environment or are expresssed intracellularly to detect typical intracellular pathogens such as viruses. Research into patterns that can consistently enhance innate immune responses and thus act as adjuvant and have potential for boosting responses in vivo, often concentrate on patterns for TLR3 receptors; assumed to recognize viral RNA, patterns for TLR5 receptors; assumed to recognize bacterial flagellin found on motile bacteria, or patterns for TLR9 receptors; assumed to recognize repetitive motifs in bacterial DNA. Possibly, (synthetic) TLR stimulants could be efficacious adjuvants especially when used in optimal combinations with well-known protective antigen(s). A new and highly-exciting approach is to fuse antigen to a TLR stimulant and in this way enhance immune responses to antigens which normally are poorly immunogenic. Indeed, TARGETFISH reported the first highly-promising results on using bacterial flagellin as a TLR stimulant and novel adjuvant to enhance immunity against a bacterial pathogen of trout for future development of adjuvants for fish vaccines.

Successful vaccines can teach us something about essential and protective immune responses

Adaptive immune responses by definition lead to an increasingly efficacious recognition of specific antigens (e.g. proteins of pathogens) based on the progressive development of immunological memory. This adaptive response forms the basis of any form of vaccination in animals including fish. As such, detailed analyses of the underlying adaptive/protective immune responses to vaccines that have previously proven to successfully protect fish from disease can help to dissect the immunological basis of memory, still largely unresolved for fish. Once dissected, the newly-acquired knowledge on immunological memory can be applied to analyze protective immune responses induced by novel, experimental prototype vaccines. Such detailed analyses are best achieved by studying well-established fish vaccines that can provide (near to) 100% protection, for example injection vaccinations of rainbow trout against viral haemorrhagic septicaemia virus (VHSV) based on plasmid DNA but also immersion vaccinations against the enterobacterium Yersinia ruckeri (causing enteric redmouth, ERM) based on inactivated bacteria and their products; two examples of existing and successful vaccine approaches building on two principly different methods and delivery routes (Figure 3).

Figure 3. Overview of different vaccine types, experimental or not, available for vaccination against viral diseases of fish.

Indeed, following immersion vaccination against Yersinia ruckeri bacteria, TARGETFISH could show the presence of long-lived B lymphocytes (bearing immunoglobulin (Ig)M on their surface) in the spleen of rainbow trout vaccinated against ERM, hinting at a possible location of immune memory in the fish spleen. At the same time, the presence of long-lived cytotoxic T lymphocytes could not be shown. Of course, T-cell receptors differ from B-cell receptors in an important way: T-cell receptors do not recognize and bind antigen directly, but instead recognize short peptide fragments bound to glycoproteins of the major histocompatibility complex (MHC) on the surface of infected or antigen-presenting cells. Thus, T-cell receptors recognize features of both the peptide antigen and the MHC molecule to which they are bound. This may have been an important factor in our observations and may help explain why a link with long-lived B lymphocytes was most clear. Of continuing interest for future analysis of protective immune responses to fish vaccines is that we were able to detect common responses of B lymphocytes after DNA vaccination with the G protein of the VHS virus. These common B lymphocyte responses indicate the presence of major populations of B lymphocytes with particular variable regions in their immunoglobulin molecules as antigen-binding sites in protected fish. Such information might give clues on which B cell responses to measure in future efficacy trials for novel vaccines.

When DNA vaccines are considered safe for fish

Traditionally, many fish vaccines have been based on inactivated bacteria or viruses, and although several of these have been extremely (cost) effective, not all diseases caused by fish pathogens can be prevented with these relatively simple forms of vaccines. DNA vaccines are considered a new approach to fish vaccination and typically rely on the administration of a DNA sequence coding for the target antigen. After injection, the host tissue cells (usually muscle cells) will take up the DNA vaccine (usually delivered by a bacterial plasmid) and express the relevant protein. The fish host will recognize this protein as non-self and mount a protective immune response through stimulation of both innate and adaptive immune mechanisms (Figure 4). Also known as genetic immunization, this method proved more effective in fish than reported in most mammalian studies and can be used without conventional oil-based adjuvants, preventing potentially unpleasant side-effects such as inflammation or adhesions often induced by oil-based adjuvants. Efficiently working DNA vaccines are able to trigger both innate and adaptive arms of the immune system. Also, because the antigen is produced by the fish cells themselves, and processed as well as exposed in the context of the correct glycoprotein (MHC) receptors on the cell surface, DNA vaccines trigger both humoral and cell-mediated immune responses. Moreover, DNA vaccines are considered relatively easy to produce and are stable at long-term storage,which are practical considerations of importance to vaccine development.

Figure 4. Immune responses triggered by DNA vaccination of fish

Although DNA plasmids do not replicate in host cells, public perception and misunderstanding of the concept of these forms of vaccines as well as fears about them leading to genetically modified organisms (GMOs) have shaped the discussion in Europe on the use of DNA vaccines. DNA vaccines are generally not considered to result in GMOs, unless the plasmid was specifically engineered to integrate and maintain genomic integration. Genomic integration of the palsmid DNA should be considered a random occurrence and would not present any additional risk as compared to e.g. the rate of natural spontaneous mutation. Yet, objective information on the degree of genomic integration of plasmid DNA after injection vaccination of fish was missing at the start of the project, although considered highly valuable for discussions on future applications of this form of vaccination. Stimulated by the DNA vaccine guidance, TARGETFISH addressed experimentally controlled studies on genomic integration of plasmid DNA after intramuscular injection of a DNA vaccine. Such information should be of value to the European authorities in charge of legislation and vaccine registration and help decide on the immediate and long-term future of DNA vaccines for fish.

The approach of TARGETFISH included the design of an experimental DNA vaccine based on the structural protein of subtype 1 of Salmon Pancreatic Disease Virus (SPDV) to study genomic integration in muscle from DNA-vaccinated Atlantic salmon collected for several days post-vaccination at the site of intramuscular (i.m.) injection. To differentiate between free and integrated plasmid only high molecular weight genomic DNA was purified. High-throughput Illumina sequencing subsequently helped to identify and count sequences with both salmon genome and plasmid DNA and estimate integration rate. Samples from muscle were calibrated against genomic DNA from a recombinant cell line carrying a known genomic integration. Further, to evaluate the limit of detection, genomic DNA from unvaccinated salmon muscle tissue was mixed with increasing amounts of DNA from the recombinant salmonid cell line with the DNA vaccine construct integrated into its genome. This controlled experimental approach mimicked different levels of genomic integration and was used to establish a calibration curve. In summary, the purification of high molecular weight genomic DNA excluding free plasmid followed by an enrichment step for fragments containing the DNA vaccine sequence and subsequent deep sequencing, helped to identify and count sequences with both salmon genome and plasmid DNA to estimate the genomic integration rate using the calibration curve. This thorough approach to evaluate genomic integration after DNA vaccination will be useful long after TARGETFISH is finished.

Practical considerations for DNA vaccination of fish

Some of the most efficient DNA vaccines described to date are those against fish rhabdoviruses. By mediating expression of the viral glycoprotein (G) in vaccinated fish, these vaccines induce rapid and long-lasting protection against the respective viruses, not only in experimental settings. Already, a DNA vaccine known as Apex-IHN has been used commercially for protection of sea-reared Atlantic salmon against infectious haematopoietic necrosis (IHN) in British Columbia (Canada) since 2005 and to date, no outbreaks of IHN have been reported among the vaccinated fish. The related disease in European rainbow trout called viral haemorrhagic septicaemia (VHS) may be historically associated mostly with aquaculture of freshwater salmonids in Western Europe, but to date is recognized to affect over 80 freshwater and marine fish species worldwide. The advantages of DNA vaccines for VHS are multiple and include i) high safety – no risk of disease, ii) rapid and long-lasting protection, iii) no additional adjuvant needed, iv) high stability and v) simple to produce. Given the present discussion on legislating DNA vaccines for use in fish, commercialisation of DNA vaccines against VHS may become a realistic option in the near future. Of course, there will be local restrictions related to the disease status: in some countries the virus has been eliminated from the freshwater by a.o. replacing earthen ponds for intensive recirculation systems and stamping out procedures, although VHSV is often still present in the marine environment. In Europe in fact, VHS is a notifiable disease and falls under the Council Directive that says that Member States shall ensure that vaccination is prohibited in any parts of their territory declared free of VHS, or is covered by a surveillance programme. Despite this Directive, Member States may allow vaccination in parts of their territory not declared free from VHS, or where vaccination is a part of an eradication programme. There is ample experimental evidence of the effectiveness of DNA vaccination against VHS, at least when based on the i.m. injection of G-protein encoding plasmids. If indeed so extremely efficient, allowing for DNA vaccination of fish against VHS can become a highly relevant new development, both in Europe or worldwide, to protect salmonids in freshwater or marine environment from this deadly disease.
Despite the recent advances made for commercialization of DNA vaccines for fish, there remain several key regulatory issues to be further explored; not only the issue of genomic integration but also determination of risk to the environment, along with practical challenges like exact plasmid vector design will remain key issues for future research. But, in general, the regulatory requirements and extent of data needed to support an application for registration of a DNA vaccine are strongly reduced when the application qualifies for use in minor (fish) species, or disease in major (fish) species which are of minor importance, or have a limited market (MUMS). This fact might speed up the commercialization of DNA vaccines for use in aquaculture, although in the light of a growing importance of aquaculture and animal welfare connected with disease outbreaks and their eradication, it cannot be excluded that the MUMS issue for farmed fish might be revised again in the future. But at this moment, with a DNA vaccine for Atlantic salmon against SPDV recently (2017) authorised for use in the EU, the future of DNA vaccination for disease prophylaxis in farmed fish appears bright.

Oral delivery of DNA vaccines

Several (experimental) DNA vaccines when injected i.m. can protect very well against a subsequent lethal challenge and provide values for relative percentage survival (RPS) >90% for months after a single administration of plasmid. Often, a local inflammatory response in the muscle tissue is observed immediately after injection and may be responsible for triggering a first protective period of innate immunity, followed by a highly specific response after a few weeks (exact timing is dependent on the temperature) correlating with occurence of specific antibodies induced locally and/or systemically (often immunoglobulin type IgM). Of specific interest are attempts to protect fish against disease using an oral rather than i.m. injected DNA vaccine, with successes reported for infectious pancreatic necrosis virus (IPNV) in rainbow trout. Here, high protection with RPS>85% were obtained, albeit based on much higher dosages of plasmid than needed for i.m. vaccination. These data suggest that DNA vaccination by oral route could become a reality in the near future. Yet, studies part of TARGETFISH indicated that the same G-protein based DNA vaccines proven fully protective when injected i.m. appear to provide little protection when applied orally despite active transcription of the G protein and induction of local immune responses in the gut. Possibly, the optimal route for induction of protection by DNA vaccines could be linked to the precise nature of the related pathogen, including parameters such as the natural site of entry and systemic versus local propagation of infection. Further studies are required to clarify these aspects of oral delivery of DNA vaccines and determine how several parameters such as encapsulation method, adjuvant effect, vaccine concentration and delivery regime might require optimization. This puts forward a plethora of research questions on oral vaccination of fish to be addressed in the near future.

Oral delivery of fish vaccines is here to stay

In mammals it is well known that antigen administration at mucosal surfaces, including oral delivery can efﬁciently trigger local as well as systemic humoral and cell-mediated immune responses. However, most of these vaccines are composed of live attenuated pathogens with associated risks such as reverting to virulence or do not trigger a long-lasting protection, thus requiring a second booster vaccination after the ﬁrst (priming) administration. Yet, a potentially great advantage of oral delivery, but also immersion delivery of vaccines to fish remains the possibility for mass vaccination based on reduced labour, expenses and maybe most important, stress effects usually associated with injection vaccination routes. On the other hand, the design of effective oral vaccines for fish are hampered by the potential breakdown of vaccine antigen if not protected sufficiently from damage in the gastro-intestinal digestive tract. It is generally presumed very important that the vaccine antigen reaches immune cells in the intestine in more or less unaltered form. In mammals, it is known that so-called “M cells” play a key role in this protective mechanism by exposing local immune cells present in the lamina propria of the gut to the vaccine antigens composing the oral vaccine. In fish, M-like cells with staining patterns similar to mammalian M cells can be found in the mucosal folds (Figure 5), suggesting the major (immune) players for antigen uptake exist in the fish gut and that all the prerequisites for successful oral vaccination protocols do exist in fish.

Figure 5. Induction of antibody (immunoglobulin, Ig) responses in the gut of fish. Scheme of a typical intestinal villus and presence of IgT+ and IgM+ B lymphocytes that, in response to oral vaccination, may be transported into the mucus layer (reproduced with permission).

One way of achieving protection against breakdown in the gastro-intestinal (digestive) tract is to coat vaccine antigens with biocompatible materials such as liposomes, ‘Antigen Protecting Vehicle’, MicroMatrix™, alginates and more until they can reach immune cells in the last part of the gut unaltered. Alginates typically have a high loading capacity and stability, and preserve well the encapsulated proteins. Another system of interest that combines aspects of correct protein folding, protection against degradation and adjuvant effects is yeast (Pichia pastoris) based antigen expression. Often, the yeast cell wall may make fragile antigens less vulnerable to degradation in the digestive tract and decrease the need for further encapsulation, while simultaneously acting as natural adjuvant. This system has been scrutinized by TARGETFISH and found suitable for both small- and large-scale production of antigens for fish vaccines because Pichia can not only grow to very high cell densities, but can also produce recombinant proteins of better quality than can bacteria and at very high protein yields. In TARGETFISH, the yeast system was explored for successfull production of Virus-Like Particles (VLPs); multimeric protein complexes whose shape mimic naturally-occurring virus particles and are highly immunogenic, but not infectious. Overall, results obtained during the TARGETFISH project suggest that oral vaccine formulations often induce local immune responses against the studied pathogens. It would appear that antigen delivery via the oral route can be an effective and promising approach to mass vaccinate fish, especially when administered at repeated intervals.

Testing vaccine efficacy, reducing animal use

At present, testing the efficacy of fish vaccines relies on standardised in vivo disease challenge models that closely mimic the natural exposure to the pathogen. This ensures that the adequate immune mechanisms necessary to control the pathogen of interest have been triggered. However, these in vivo challenges typically require relatively large numbers of fish to be challenged and thus are expensive and animal unfriendly. Clearly, reliable in vitro alternatives for monitoring vaccine efficacy and testing batch potency would imply a major step forward. Ideally, methods such as enzyme-linked immunosorbent assays (ELISAs) are available to detect the different antibody types in serum and/or mucus samples opening up in vitro alternatives for in vivo challenges.

Also in fish, antibodies (immunoglobulins) are thought to play crucial roles in adaptive immune responses after vaccination, particularly against bacterial antigens, by recognizing the pathogen and helping with its destruction through various processes such as complement activation and maybe also phagocytosis. Fish B lymphocytes express different and mutually exclusive immunoglobulin heavy chains and as a result can secrete different types of antibodies (for example IgM, or IgT). IgM is the most common immunoglobulin found in both serum and mucus and is the key player in systemic immune responses, whereas IgT is considered the main responder in mucosal surfaces. Similar to IgM responses in fish serum which are generally easy to quantify and sometimes predictive for protection, it remains likely that specific IgT levels in mucus may act as an indicator of vaccine efficacy testing for mucosal (immersion, oral) vaccines. Detection of IgT gene expression through PCR and immunohistochemistry already have proven successful in tissue samples following immersion vaccination. In such cases specific antibody responses can be used as a first indicator of vaccine efficacy or batch potency testing. In line with the expectation that mucosal vaccination routes (immersion, oral) will keep finding their way to aquaculture practise, even though routine measurements of specific IgT in serum and mucosa remain a technical challenge, it makes sense to keep examining the use of mucosal IgT as indicator of oral vaccine efficacy. In summary, although antibody responses in fish maybe are less impressive with regard to properties of memory when compared to the logarithmic increases of antibody affinity seen in mammals, and although clonal expansion of B lymphocytes in fish may not always be extremely obvious, nor are adaptive antibody responses particularly fast, fish can be protected for long periods and thus vaccination of fish clearly remains the way forward.

Further scientific reading

1. Developmental and Comparative Immunology 2014: Special Issue on Immunity to Infectious Diseases of Fish
2. Frontiers in Immunology 2015-2016: Research Topic on Immunotherapies targeting fish mucosal immunity - Current knowledge and future perspectives
3. Developmental and Comparative Immunology 2016: Special Issue on Intestinal Immunity
4. Fish and Shellfish Immunology 2018: Special Issue on Targeting Fish Vaccination
5. Bulletin of the European Association of Fish Pathologists 37 (7), 2018
6. www.targetfish.eu

Potential Impact:
With an average annual growth rate worldwide of 7% aquaculture remains the fastest growing animal food production sector and a crucial source of high-quality protein for human consumption. Aquaculture currently provides about half of the worldwide seafood supply for human consumption, with potential for further growth (FAO). Aquaculture production in Europe provides employment to 100,000 people, generating an annual turnover of 7 billion EURO and the EU is responsible for one-fifth of the global production. Yet, partly due to a lack of authorised veterinary products for medicinal treatment, outbreaks of disease in farmed fish species continue to cost the sector up to 20 % of its production value. The most appropriate method for controlling the spread of disease is to prevent it from starting in the first place, through vaccination.

Over the last few decades vaccine developments have already contributed in reducing diseases outbreaks and fish mortalities. Despite this progress, mainly because of lack of sufficient knowledge base and of veterinary medicinal products authorised for use, at the start of TARGETFISH fish diseases still impeded a further sustainable development of European aquaculture. Excellence in research and innovation should lead to optimal health of cultured aquatic animals and underpin better performance and competitiveness of European aquaculture, as highlighted by the European Commission and supported by statements of the European Aquaculture and Technology Innovation Platform (EATiP). TARGETFISH aimed to reduce the impact of fish diseases on the European aquaculture industry and strived to advance the development of existing but not sufficient and new prototype vaccines against several socio-economically important viral or bacterial pathogens.

TARGETFISH aimed to provide a long-lasting contribution to the prevention of important fish diseases in the European aquaculture industry by developing fish vaccines against several important bacterial and viral pathogens with serious economic impact. Specifically, excellent research and innovation focused on understanding protective immunity by studying the relevant protein antigens from several fish bacteria and viruses, new oral (mass vaccination) systems for delivering these antigens to mucosal body sites, and new adjuvants for improving the duration of immunity following fish vaccination. This information was then evaluated against both mucosal and systemic protective immune responses to develop read-outs for vaccine efficacy. Researchers next turned their attention to learning from and improving upon, existing vaccines and focused on prototyping vaccines whose efficacy was then validated via in vitro assays and in vivo challenges. Successful vaccines were evaluated for minimal side effects and maximum safety. Lastly, vaccination protocols were scrutinised against field conditions, shortening the route for the implementation of the project’s results. These results were then presented to policy makers, scientists and industry leaders and in some cases, taken up for further vaccine development.

TARGETFISH has been a multidisciplinary project taking off with 30 partners from 10 EU member states, 2 associated countries and 1 International Cooperation Partner Country (ICPC), with 16 academic (Research, Technology & Development, RTD), 12 SME (Small - Medium Enterprises) and 2 IND (Industrial) partners forming a large collaborative consortium. From its start TARGETFISH carefully considered this a suitable partnership composition that would ensure industry relevance through continuous dialogue, leading to a most successful innovation lifecycle for vaccine development. This project structure facilitated close interaction between academia and industry, reflecting the joint decision that the project should have representation from companies involved in all stages of the process, with the final consortium spanning a more-or-less equal division between public research institutions and European industries.

TARGETFISH addressed, in a balanced effort, the six major farmed fish species in Europe, including Atlantic salmon as the most cultivated fish species in Europe and rainbow trout, common carp as one of the most cultured fish species for food consumption in Central Europe, and turbot, European sea bass and gilthead seabream as the most important farmed marine species in Southern Europe. An Industry Forum was designed as a suitable interface for interaction between academia and industry, initially with a focus on research and development, later as a true communication platform to disseminate knowledge outputs. In this way, TARGETFISH not only united world-leading scientists with a complete understanding of the protective immune pathways triggered by effective vaccines but - at the same time – made sure to consult with the industry actors to study and improve mass delivery methods and vaccine strategies of farmed fish.

The Industry Forum provided a platform for a continuing validation and communication of the applied potential of the research outcomes to those not always directly involved with the project but interested in the fish vaccine market. Thereby, the Industry Forum, via joint participation of TARGETFISH partners and vaccine industry revolutionised the concept of fish vaccine development. At three consecutive meetings during the international meetings of the European Association of Fish Pathologists (EAFP) in Tampere, Finland (2013), Las Palmas, Spain (2015), and Belfast, Northern Ireland (2017) the TARGETFISH Industry Forum presented and discussed market applicability of improved vaccines and new prototype vaccines that came forward from the project. Three representative examples of prototype vaccine approaches, all technically different but all with high socio-economic impact and commercial potential are highlighted below.

Vaccinating rainbow trout against fry syndrome

Rainbow trout fry syndrome (RTFS) is caused by the Gram-negative bacterium Flavobacterium psychrophilum which has been responsible for substantial economic losses in the rainbow trout industry globally and for decades. The disease is widespread, occurs frequently, and can cause high mortality in fry freshwater hatcheries but also larger fish in on-growing sites. An average early-stage mortality of 10% in rainbow trout could be considered normal in many production sites. Disease episodes tend to occur between 10-14 °C with skin lesions surrounding the dorsal fin and tail, while in very small fish no clinical signs are apparent and death occurs due to systemic infection. The impact of RTFS can be seen in terms of economic cost and in terms of risk and antibiotics are often used to treat affected stock. In fact, without vaccine, the only course of action is antibiotic treatment, which use led to increased levels of antibiotic resistance, highlighting the urgent need for a vaccine against RTFS.

TARGETFISH developed a prototype vaccine against RTFS. The diversity of the bacterial isolates responsible for the disease and the inherent difficulties in vaccinating juvenile fish hampered for a long time the development of a vaccine. The efficacy of a new immersion vaccine for RTFS was tested in large-scale trials by immersion challenge against two heterologous bacterial isolates which showed promising relative percent survival (RPS) values of >50%. These values were recognized as an essential step forward to submit experimental field trial protocols and field-test small quantities of RTFS vaccines, manufactured under good manufacturing practise (GMP), using both injection and immersion routes. Pending successful demonstration in the field, market authorization is only a small step away.

Vaccinating sea bass against viral encephalopathy and retinopathy

Turbot, gilthead seabream and European sea bass are the most important farmed marine species in Southern Europe. Although the production of sea bass has increased continuously, a big threat to sustainable sea bass farming remains its sensitivity to viral nervous necrosis (VNN), a serious disease induced by a Nodavirus. A major concern is that viral encephalopathy and retinopathy virus (VERV) strikes early in the lifecycle of the fish (larval or fry stages), before current protocols mae possible vaccination.

TARGETFISH developed a prototype vaccine against VERV based on Virus-Like Particles (VLP) produced in the yeast Pichia pastoris by expression of virus-derived capsid protein genes in shake flask cultures or lab-scale fermenters. The prototype vaccine showed high immunogenicity in sea bass and the first attempts to protect sea bass by oral vaccination with crude recombinant yeast extract containing VERV-VLP gave promising results. A subsequent comparison of intra-peritoneal (IP) injection versus oral delivery of the vaccine at laboratory scale followed by a lethal challenge, showed relative percent survival (RPS) values that were equal after oral and injection vaccination. Clearly, purified VLPs provide a potent source of protective proteins for vaccination of sea bass against VERV and the prototype recombinant sea bass nodavirus (VERV) VLP-based vaccine provides a first step towards commercial application. Up-scaling production in yeast and initiating testing under field conditions is now considered the necessary next step to demonstrate the applied potential of this next generation fish vaccine.

Vaccinating against salmon pancreatic disease

Salmon Pancreatic Disease (SPD) is a serious infectious disease which causes damage to the heart, pancreas and skeletal muscle and can lead to the death of salmon. The disease has become established in some Member States and outbreaks of SPD cause significant losses in salmon farms in the EU. Recently (2017) a DNA vaccine for Atlantic salmon against Salmon Pancreatic Disease (SPD) caused by subtype 3 of the SPDV virus was authorised for use in the EU. The authorisation was facilitated by the rule that the data requirements for authorisation of a (DNA) vaccine are reduced when a vaccine classifies as ‘intended for minor use or for the minor species (MUMS)/limited market’. Data requirements for authorisation of a new (DNA) vaccine include safety studies carried out in the target species with a vaccine dose that is recommended for use and of course, the product should be produced according to the manufacturing process. Most fish vaccines are intended for use in a minor species, or for use against a disease relevant in a major species but for minor use, or have a limited market, all classifications reducing the data requirements for authorisation of a new fish vaccine. Although the Norwegian Medicines Agency voiced some concerns against the new DNA vaccine based on the short (2 months) duration of immunity studies not outweighing the possible risks related to animal safety, potential lack of protection beyond 2 months, potential interaction with other vaccines used in Norway and potential reduction in filet quality as a consequence of intramuscular injection of the vaccine, the recent EU authorisation provides a crucial step forward to the use of DNA vaccines in aquaculture.

DNA plasmid vaccines are new innovative veterinary products for regulators. A DNA vaccine by itself cannot be considered a genetically modified organism (GMO), since a plasmid is a construct and not capable, on its own, to replicate or transfer genetic material into the genome of the fish. Therefore, a plasmid cannot be regarded as a biologically viable entity. Yet, there remains the question as to whether the fish vaccinated with the plasmid become genetically modified themselves. GMOs are defined as organisms in which the genetic material has been altered in a way that does not occur naturally by mating and/or natural recombination. Although the exact position for future DNA vaccines still remains unclear, the answer appears to be that DNA vaccines and DNA-vaccinated fish should not be considered genetically modified organisms. Yet, potential integration into the genome of the (DNA) vaccinated animal remains a critical safety issue and should be thoroughly investigated through the relevant laboratory safety studies and environmental risk assessment. The importance of objective research on this form of fish vaccination must be clear. Of course, a safety study should demonstrate a negligible risk for humans exposed to the new vaccine. Although DNA vaccines are not classified as live vaccines it is considered prudent to approach some of the safety studies required for live vaccines. An environmental risk assessment (ERA) should be included and should be extensive, depending on the potential environments the product may be used in and include a quantitative assessment with consequence analysis. The safety study could be conducted as a 10-fold overdose and should include dissemination of the DNA in the target animal with consideration for the potential integration into relevant organs and tissues including gonadal tissue.

The approach of TARGETFISH included the design of an experimental DNA vaccine based on the structural protein of subtype 1 of the SPDV virus to study genomic integration in muscle samples from DNA-vaccinated Atlantic salmon collected for several days post-vaccination at the site of intramuscular injection. To differentiate between free and integrated plasmid researchers specifically purified the high molecular weight genomic DNA fraction and then performed high-throughput next generation sequencing, which helped to identify and count sequences with both salmon genome and plasmid DNA and estimate integration rate. Samples from muscle were calibrated against genomic DNA from a recombinant cell line carrying a known genomic integration. This recombinant cell line was crucial to evaluate the limit of detection; genomic DNA from unvaccinated salmon muscle tissue was mixed with increasing amounts of DNA from the recombinant salmonid cell line with the DNA vaccine construct integrated into its genome. These data mimicked different levels of genomic integration and were used to establish a calibration curve. Overall, the purification of high molecular weight genomic DNA excluding free plasmid followed by an enrichment step for fragments containing the DNA vaccine sequence and fiannly, the deep sequencing approach, all helped to identify and count sequences with both salmon genome and plasmid DNA to estimate the genomic integration rate using the calibration curve. This strucural approach to study genomic integration after DNA vaccination will be useful long after the closure of TARGETFISH.

Dissemination and exploitation of project results

In order to maximise dissemination, knowledge transfer and exploitation of the TARGETFISH project outputs, the consortium specifically focused on bridging the gap between knowledge application and knowledge exploitation in the fish health field. This was considered a crucial element in the link between the RTD performers and the SME/Pharma industry who participated in the project. The TARGETFISH website (www.targetfish.eu) has been and will remain the natural hub to more than 100 scientific publications, close to 40 dissemination events as well as three videos on the project results or working mechanisms of fish vaccines, all three videos top-10 hits at YouTube searches based on ‘fish vaccination’.

Knowledge transfer has been an important issue for the partnership of TARGETFISH requiring tailor-made actions based on crucial steps, namely identification and analysis of the “primary user” or sometimes also referred to as “next-user(s)” of specific units or clusters of knowledge and applications. Furthermore, by carrying out a focused knowledge management approach as an integrated part of the project design, it became possible to capture several knowledge outputs, including those related to methodologies and protocols used in the project. By monitoring, collecting and managing knowledge outputs it became possible to fast track prototype vaccines for adoption by industry and therefore commercial application. One important means of managing knowledge outputs included communication with the COLUMBUS project (www.columbusproject.eu/) allowing for targeted knowledge transfer and assuring that TARGETFISH outputs have positive societal benefit. In this way, applicable knowledge generated through EC-funded science and technology research such as generated as part of TARGETFISH could be transferred effectively to advance the governance of the marine and maritime sectors while improving competitiveness of European companies and unlocking the potential of the oceans to create future jobs and economic growth in Europe (Blue Growth). Through the aquaculture node, knowledge outputs generated in the course of the TARGETFISH project were moved closer to an industry implementation phase to mature them for industrial uptake. The TARGETFISH project is very proud that is has become the only EU project - among all the EU Aquaculture funded projects - that has contributed two case studies, one on the prototype vaccine for nodavirus infections of seabass and one on automated vaccination machines teamed up to provide a vaccination service for the Mediterranean mariculture industry.

Collaboration between academia and industry pays off

TARGETFISH was built on a generic knowledge- and technology-base for the fish immune system provided by among others previous FP6-funded projects, which allowed for rapid progress on identifying key elements that determine adaptive immunity and memory and created the next level of scientific base for successful vaccination and long-term protection of fish. As such, the FP6 and FP7 projects provide very strong arguments for a form of continuing support for research relevant to the prevention of important fish diseases in the European aquaculture industry. These projects not only have provided basic immunological knowledge but also have shown a direct impact on the market applicability of several improved or new prototype vaccines, resulting in a long-lasting impact on fish vaccination. Assisted by academia in the early research and development phase, small- to medium-sized enterprizes have developed tailored products and are finding their own markets within fish vaccinology complementing those of the larger vaccine companies. Yet, the job is far from completed with just a few examples including ongoing research on new ‘molecular’ adjuvants to improve existing and novel vaccines and ongoing research on DNA vaccines for fish for which the European Medicine Agency foresees a period for continued attention and continued effort of academics. No doubt, there remains a strong need for academia and enterprises to join forces on oral delivery of fish vaccines.

Reducing environmental impact of veterinary treatments

There is growing international concern about the potential impact of antibiotic residues on the environment in general and the possibility that they increase antibiotic resistance of microorganisms. For many years already, the successful implementation of efficacious vaccines e.g. for Atlantic salmon in Norway has demonstrated the high potential of disease prophylaxis through vaccination, reducing the need for and use of antibiotics. To help safeguard the environment, reducing the amount of antibiotics needed to treat disease outbreaks, TARGETFISH contributed to the prevention of a number of impactful fish diseases in the European aquaculture industry through the use of vaccines.

Overall, TARGETFISH addressed more than 10 bacterial or viral diseases of the six major farmed species in Europe considered important based on the following criteria: i) Mortalities and direct losses due to the pathogen; ii) World Organisation for Animal Health (OIE) listing; iii) The availability (or not) of an existing vaccine and whether the vaccine could be further optimised, e.g. by an alternative administration route or adjuvants; iv) The absence of readily available alternatives to vaccination; v) Existing national and international initiatives that already address the fish disease problem to a sufficient degree; vi) Consultation with authorities in charge of approving veterinary medicines. TARGETFISH optimized a selected number of prototype vaccines, helped to demonstrate their market applicability making them of high interest to vaccine companies wishing to turn the knowledge acquired into market products as soon as possible. Lst but not least, TARGETFISH covered a broad spectrum of fish immunology and fish health, studying key pathways considered essential for the development of efficacious vaccines in fish. The outcomes of these detailed studies helped clarify important immunological issues central to the development of any future fish vaccine.

List of Websites:
www.targetfish.eu
targetfish.cbi@wur.nl

Final Report Summary - TARGETFISH (Targeted disease prophylaxis in European fish farming)

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